Download ImVacS 2012 Immunotherapeutics and Vaccine

ImVacS 2012
Immunotherapeutics and Vaccine Summit, Cambridge, MA.
Aug. 13-16, 2012
Abstract
Keyhole Limpet Hemocyanin (KLH): A Unique Unlikely Pharmaceutical Product
Chow, Herb, Sagermann, Martin and John Sundsmo. Stellar Biotechnologies, Inc., Port
Hueneme, CA. 93041.
The giant keyhole limpet Megathura crenulata, is an unlikely organism for commercial
mariculture compliant with GMP standards. Discovery by immunologists > 50 yrs ago , that the
hemocyanin oxygen-carrier protein constituted also an extremely effective antigen-carrier for
inducing immunity, heralded uses in a variety of vaccines including more than 18 active human
clinical trials for treatments of Alzheimer’s disease, autoimmune diseases and cancer. Human
uses of KLH have also included testing immune status of patients to identify primary immune
deficiency diseases. In addition, manufacturers of biopharmaceutical products have found uses
of KLH in early preclinical testing for possible untoward immune side effect , i.e., T-dependent
antigen responses (TDAR) testing. While safety of KLH in humans has been unequivocally
established in more than 40 yrs of use, remarkably little detailed information is available to
elucidate the possible KLH molecular mechanisms of action in Th1 and Th2 responses. In the
interest of better understanding KLH induced T-cell responses at a cellular immune level, we
investigated KLH TDAR antibody and cell mediated immune responses (CMI) in rabbits, guinea
pigs, mice and rats using both a proprietary purified native high molecular weight (6-8MDa)
didecameric (20-mer) KLH and our commercial GMP stable KLH subunit (dimeric; 700-800kDa)
formulation, i.e., with IgM-IgG class-switch antibody analysis ; DTH in guinea pig and rabbit;
and, cytokine and ELISpot analyses in the mouse. With expected species-specific difference in
TDAR responses, the overall findings support KLH induction of both Th1 and Th2 responses, i.e.,
MHC class-I and MHC class-II, respectively, in guinea pig and rabbit. Significant differences in
TDAR were observed between different KLH preparations that biochemically appeared identical
by PAGE, SDS-PAGE, SEC and IEC. Flow cytometric analysis supports activation of CD11+
dendritic cells, as evidenced by upregulation of CD86 co-stimulatory markers. Activated
dendritic cells, internalize and process antigen through either endocytic (C-lectin; Th1) or
lysosomal (Th2) pathways. We subjected different KLH preparations to N-glycan analysis to
elucidate structures. Consistent with the observed immune activities, KLH N-glycans contain
structural fucosylated—galactosyl-mannans capable of binding C-lectin receptors such as the
mannose receptor (ManR), Langerin receptor and DC-SIGN; and S-lectin receptors such as
galectin. Cytokine and ELISpot splenic cytokine analyses in the mouse revealed induction of IL4, IL-6, TGF-β and recruitment of IL-4 secreting T cells in both primary and secondary immune
responses. Greater recruitment of IL-4 secreting T cells in the secondary immune response was
correlated with higher levels of anti-KLH IgG in serum. Thus, KLH appears to be a uniquely well
qualified alternative to TLR-directed adjuvants for use as a carrier with adjuvant-like activity in
vaccine products, as well as, for use in patient and animal TDAR testing for emergent
pharmaceutical products.